Recombinant Protein Production
To date, more than 130 proteins with human therapeutic use have entered the market. Only a small number of proteins are expressed in their native cell type under physiological conditions in amounts that permit convenient purification of the relatively large quantities required for research and clinical use. For instance, bone contains very low amounts of native bone morphogenetic proteins (BMPs), a family of proteins members of which are used clinically to promote bone repair. There are methods that exist to extract and purify biologically active BMPs from bone, but these methods are time-consuming, labor intensive, and most importantly, result in a very low yield: starting from 15 kg raw bone, final yield is around 0.5 g of partially purified BMPs (see Urist et al. Methods Enzymol 1987; 146:294-312 and Hu et al. Growth Factors 2004; 22:29-33). Therefore, various expression systems have been developed to produce recombinant proteins. Single cell expression systems have used a variety of hosts including bacteria, baculovirus-infected insect cells, yeast, and mammalian cells.
In bacteria based expression systems most proteins are produced at a relatively large volume compared to other single cell expression systems. However, the bacterial expression system lacks the ability to modify proteins, and hence fails to generate dimerized, correctly folded, and glycosylated functional forms of the mature proteins. Extensive dimerization and renaturation processes are thus often required before the recombinant proteins can be used. (Cleland, 1993, In Protein Folding In Vivo and In Vitro: pp. 1-21). Further, the recovered recombinant protein is usually contaminated with endotoxin/pyrogen that makes proteins for pharmaceutical/diagnostic use extremely difficult to validate (Walsh and Headon, 1994, In “Protein Biotechnology”, pp. 118-162).
Recombinant DNA technology allows mammalian cells that usually grow well in culture to produce heterologous proteins, or proteins not normally synthesized by these cells. Genetic engineering allows high expression of the gene coding the protein of interest using vectors that are designed to replicate foreign DNA, and control transcription and translation of the introduced gene. Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications because of their proper protein folding, assembly, and post-translational modification (Wurm Nat Biotechnol 2004; 22:1393-1398) The quality and activity of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria and insect cells.
Mammalian expression systems are relatively costly to maintain in comparison to other expression systems and in general the amounts of protein produced are lower than in bacterial systems (for review see Wurm Nat Biotechnol 2004; 22:1393-1398). The productivity of recombinant cell lines has increased dramatically in the past 20 years. In the 1980s, mammalian cells typically reached a density of about 2×106 cells/ml with a batch process production phase of about 7 days and a specific productivity slightly below 10 pg/cell/day. In a process reported in 2004, the culture was started at a low cell density of about 100,000 cells/ml and rapidly grew into a density of more than 10×106 cells/ml. A high level of cell viability was maintained for almost 3 weeks with a specific productivity up to approximately 90 pg/cell/day (Wurm Nat Biotechnol 2004; 22: 1393-1398). The high yield obtained in today's processes are the result of years of research that led to a better understanding of gene expression, metabolism, growth and apoptosis in mammalian cells. Overall efforts have led to improvements in vectors, host cell engineering, medium development, screening methods, and process engineering and development.
Other single cell expression systems such as insect and fungal expression systems have also been used for recombinant protein production. However, these expression systems are considered to suffer from similar problems as does the bacterial expression system (misfolding, improper processing) (Martegani et al., Appl. Microbiol. Biotechnol. 1992; 37:604-608). Recombinant proteins expressed by insect cells are often glycosylated incompletely or have different glycosylation patterns from those produced by mammalian cells. Some strains of yeast cells cannot perform N-linked or O-linked glycosylation or both (for a review of insect cell culture, see Goosen, et al., Insect Cell Culture Engineering. New York: M. Dekker, 1993, and for yeast expression system, see Chiba and Jigami Curr Opin Chem Biol. 200; 11:670-676).
Besides single cell systems, multi-cellular organisms such as transgenic plants or animal have been used for transgenic protein production.
Disadvantages of transgenic plants include low accumulation level of recombinant protein, insufficient information on post-translational events (e.g., unknown glycosylation pattern), and the lack of data on downstream processing (for reviews see Boehm, Ann. N.Y. Acad. Sci. 2007: 1102; 121-134, Horn et al. Plant Cell Rep. 2004: 22; 711 and Kusnadi et al. Biotechnol. Bioeng. 1997: 56; 473-484).
One major concern with transgenic animals is the possibility of disease transmission from animal to human. Other challenges such as inefficient introduction of foreign DNA into host animal and gaps in our knowledge of embryo genomics and epigenetic changes need to be overcome in order to optimize the transgenic animal systems for recombinant protein production (for reviews see Niemann and Kues Reprod. Fertil. Dev. 2007: 19; 762-770; Velander et al. Scientific American 1997: 276; 70-74, Pollock et al. J. Immunol. Methods 1999: 231; 147-157).
Bone Morphogenetic Proteins
The bone morphogenetic proteins (also called bone morphogenic proteins or BMPs) are members of the transforming growth factor beta (TGFβ) superfamily of secreted growth and differentiation factors. The BMP subfamily of the TGFβ superfamily comprises at least fifteen proteins, including BMP-2, BMP-3 (also known as osteogenin), BMP-3b (also known as growth and differentiation factor 10, GDF-10), BMP-4, BMP-5, BMP-6, BMP-7 (also known as osteogenic protein-1, OP-1), BMP-8 (also known as osteogenic protein-2, OP-2), BMP-9, BMP-10, BMP-11 (also known as growth and differentiation factor 8, GDF-8, or myostatin), BMP-12 (also known as growth and differentiation factor 7, GDF-7), BMP-13 (also known as growth and differentiation factor 6, GDF-6), BMP-14 (also known as growth and differentiation factor 5, GDF-5), and BMP-15 (for a review, see e.g., Azari et al. Expert Opin Invest Drugs 2001; 10:1677-1686).
BMPs are synthesized as large precursor molecules consisting of an amino terminal signal peptide, a pro-domain, and a carboxy terminal domain harboring the mature protein. The amino-terminal signal peptide and pro-domain regions of the various BMPs vary in size and amino acid sequence, whereas the mature domain shows a greater degree of sequence identity among BMP subfamily members. The mature domain is ordinarily cleaved from the pro-domain by one or more of the basic proprotein convertases, such as furin, to yield an active mature polypeptide of between 110-140 amino acids in length. The pro-domain appears to be required for normal synthesis and secretion of BMP polypeptides (for a review, see e.g., Azari et al. Expert Opin Invest Drugs 2001; 10:1677-1686; and Hoffman et al. Appl Microbiol Biotech 2001; 57:294-308, Israel et al. Growth Factors 1992; 7:139-150).
The individual members of the BMP family can be divided into several subfamilies within which the sequence of their mature carboxy terminal protein domain is well conserved. BMP-2 and -4 have greater than 90% sequence identity and BMP-5, 6, 7 and 8 have 70 to 90% sequence identity within these subfamilies. Between these 2 groups there is a 55 to 65% sequence identity of the mature proteins. In contrast the mature forms of the TGF-β, the activin and the inhibin families share less that 50% sequence identity with these BMPs (Ozkaynak et al. J Biol Chem. 1992; 267:25220-25227).
The highly conserved mature region of BMPs contain seven highly conserved cysteine residues. Six of these cysteine residues are implicated in the formation of intrachain disulfide bonds that form a rigid “cysteine knot” structure. The seventh cysteine is involved in the formation of homodimers and heterodimers via an interchain disulphide bond (for a review, see e.g., Azari et al. Expert Opin Invest Drugs 2001; 10:1677-1686 and Hoffman et al. Appl Microbiol Biotech 2001; 57:294-308).
During intracellular processing, the mature domain of BMPs are cleaved from the pro-domain. The mature BMP polypeptides form either homodimers (made up of monomers of a single BMP subfamily member) or heterodimers (made up of monomers of two different BMP subfamily members) connected by one disulfide bond in a head-to-tail arrangement (for a review, see e.g., Azari et al. Expert Opin Invest Drugs 2001; 10:1677-1686 and Hoffman et al. Appl Microbiol Biotech 2001; 57:294-308). Both BMP homodimers (e.g., BMP-2/-2 homodimers) and heterodimers (e.g., BMP-4/-7 heterodimers) are active in vivo (see, e.g., Aono et al. Biochem Biophys Res Comm. 1995; 210:670-677; Kusumoto et al. Biochem Biophys Res Comm 1997; 239:575-579; and Suzuki et al. Biochem Biophys Res Comm 1997; 232:153-156). Under certain conditions, heterodimers of BMP-2, BMP-4, and BMP-7 (e.g., BMP-4/-7 heterodimers and BMP-2/-7 heterodimers) are more active oseoinductive agents than the corresponding homodimers (see, e.g., U.S. Pat. No. 6,593,109 and Aono et al. Biochem Biophys Res Comm. 1995; 210:670-677).
BMPs are glycosylated proteins, with the mature protein having between 1 and 3 potential glycosylation sites (Celeste et al. PNAS 1990; 87:9843-9847). A glycosylation site in the center of the mature protein domain is shared by BMPs 2, 4, 5, 6, 7, and 8 but is absent in BMP-3 (Ozkayanak et al. J. Biol. Chem. 1992; 267:25220-25227). Chemical deglycosylation of BMP-2 and BMP-7 results in reduced activity of these proteins (Sampath et al. J. Biol. Chem. 1990; 265:13198-13205), indicating that proper glycosylation is required for full BMP activity.
Active, mature BMP polypeptides bind to, and initiate a cell signal through, a transmembrane receptor complex formed by types I and II serine/threonine kinase receptor proteins. Type I (BMP receptor-1A or BMP receptor-1B) and Type II (BMP receptor II) receptor proteins are distinguished based upon molecular weight, the presence of a glycine/serine-rich repeat, and the ability to bind to specific ligands. Individual receptors have low affinity binding for BMPs, while heteromeric receptor complexes bind to BMPs with high affinity (for a review, see e.g., Azari et al. Expert Opin Invest Drugs 2001; 10:1677-1686 and Hoffman et al. Appl Microbiol Biotech 2001; 57:294-308).
BMPs have been shown to regulate the growth and differentiation of several cell types. They stimulate matrix synthesis in chondroblasts; stimulate alkaline phosphatase activity and collagen synthesis in osteoblasts, induce the differentiation of early mesenchymal progenitors into osteogenic cells (osteoinductive), regulate chemotaxis of monocytes, and regulate the differentiation of neural cells (for a review, see e.g., Azari et al. Expert Opin Invest Drugs 2001; 10:1677-1686 and Hoffman et al. Appl Microbiol Biotech 2001; 57:294-308).
One of the many functions of BMP proteins is to induce cartilage, bone, and connective tissue formation in vertebrates. The most osteoinductive members of the BMP subfamily are BMP-2, BMP-4, BMP-6, BMP-7, BMP-9, and BMP-14 (see, e.g., Hoffman et al. Appl Microbiol Biotech 2001; 57-294-308, Yeh et al. J Cellular Biochem. 2005; 95-173-188 and Boden. Orthopaedic Nursing 2005; 24:49-52). This osteoinductive capacity of BMPs has long been considered very promising for a variety of therapeutic and clinical applications, including fracture repair; spine fusion; treatment of skeletal diseases, regeneration of skull, mandibullar, and bone defects; and in oral and dental applications such as dentogenesis and cementogenesis during regeneration of periodontal wounds, bone graft, and sinus augmentation. Currently, recombinant human BMP-2 sold as InFUSE™ by Medtronic and recombinant human BMP-7 sold as OP-1® by Stryker are FDA approved for use in spinal fusion surgery, for repair of fracture non-unions and for use in oral surgery.
Other therapeutic and clinical applications for which BMPs are being developed include; Parkinson's and other neurodegenerative diseases, stroke, head injury, cerebral ischemia, liver regeneration, acute and chronic renal injury (see, e.g., Azari et al. Expert Opin Invest Drugs 2001; 10:1677-1686; Hoffman et al. Appl Microbiol Biotech 2001; 57:294-308; Kopp Kidney Int 2002; 61:351-352; and Boden. Orthopaedic Nursing 2005; 24:49-52). BMPs also have potential as veterinary therapeutics and as research or diagnostic reagents (Urist et al. Prog Clin Biol Res. 1985; 187:77-96).
Production of Recombinant BMPs
The widespread therapeutic use of BMPs has been hindered by difficulties in obtaining large quantities of pure, biologically active BMP polypeptide, either from endogenous or recombinant sources at a cost-effective price. As noted above bone and other tissues contain very low concentrations of mature BMPs and BMP precursor molecules. While methods exist to extract biologically active BMPs from bone, these are time consuming methods with non-economical yields (Hu et al. Growth Factors 2004; 22: 29-33).
Recombinant BMPs have been produced using bacterial expression systems such as E. coli. However, active BMPs are obtained only following an extensive renaturation and dimerization process in vitro. In this process, monomeric BMP must first be purified, then renatured in the presence of chaotropic agents, and finally purified to remove unfolded BMP monomers and other contaminating E. coli proteins. This process is complex, time consuming, and costly, and often has a low yield of active dimer compared to total monomer produced (for a review, see e.g., Hoffman et al. Appl Microbiol Biotech 2001; 57:294-308). Furthermore, BMPs produced by such methods are not glycosylated, and therefore would not be expected to be fully potent.
Attempts at recombinant production of BMP in insect cell culture have resulted in predominantly intracellular BMP accumulation with minimal recovery of active BMP from the culture media (Maruoka et al. Biochem Mol Biol Int 1995; 35:957-963 and Hazama et al. Biochem Biophys Res Comm 1995; 209:859-866).
Commercially available BMP preparations are based upon mammalian cell expression systems. Human BMP-2 has been expressed in CHO (Chinese hamster ovary) cells; human BMP-4 has been expressed in a mouse myeloma cell line (NS0) and in a human embryonic kidney cell lines (HEK 292); and human BMP-7 has been expressed in a primate cell line (BS) and in CHO cells (for a review, see e.g., Hoffman et al. Appl Microbiol Biotech 2001; 57:294-308). However, such eukaryotic expression systems generally have lower productivity and yield compared to prokaryotic systems. Further, BMPs appear to be produced inefficiently in eukaryotic systems resulting in much lower levels of production compared to that achieved for other proteins in the same systems (Israel et al. Growth Factors 1992; 7:139-50). Due to these low yields, recombinant BMPs are currently very expensive.
Thus, a need exists in the art for materials and methods for the production of recombinant, active BMPs on a large scale. In particular, a need exists for materials and methods for efficient, lost-cost production of biologically potent BMPs.
Current Methods to Enhance Recombinant Protein Production
Efforts to improve productivity in mammalian cell systems can be divided into 2 areas. The first involves increasing or maintaining transcription of the transgenes by methods such as improving vector design, plasmid integration and optimizing the chromosomal environment. The second is maximizing the translational or secretory capacity of the host cells through methods such as host cell engineering, media optimization, and improved bioreactor design and feeding methods (for review see Wurm Nature Biotechnol. 2004; 22:1393-1398, Barnes & Dickson Curr. Opin. Biotechnol. 2006; 17:381-386).
Many stimulating chemicals have been added to the culture systems to improve productivity. Examples include butyrate (Lamotte et al. Cytotechnology, 1999; 29: 55-64), which enhances gene expression by inhibiting histone deacetylases, pentanoic acid (Liu et al. J. Biosci. Bioeng. 2001; 91: 71-75) and cysteamine (Yoon et al. Biotechnol. Lett. 1999; 20: 101-104).
Role of Pro-Domains in Protein Processing and Secretion
Many proteins including those with therapeutic applications are produced in nature as pro-proteins. Pro-proteins are larger precursors of the mature protein. The pro-protein consists of the pro-domain and the mature domain. The pro-domain of a protein plays an important role in the processing and secretion of the mature protein. The best understood role for a pro-protein is that derived from studies of pro-hormones and pro-enzymes, where cleavage is associated with the mature protein activation. Pro-forms of growth factors have received intensive scientific attention recently because pro-domain is found to play essential roles in the maturation of the precursor proteins.
Studies on the role of the prodomain of many diverse proteins have demonstrated that they play a role in the processing and secretion of these proteins. The prodomain of brain derived neurotrophic factor (BDNF) interacts with sortillin an intracellular chaperone which controls the sorting of BDNF to the regulated secretory pathway. A single amino acid mutation in the prodomain results in defective regulated secretion of BDNF by altering its interaction with sortillin (Chen et al. J. Neuroscience 2005; 25:6156-66). The prodomain of Conotoxin-TxVI shields the hydrophobic surfaces of the mature protein, which would otherwise target it for intracellular degradation, permitting its secretion (Conticello et al. J. Biol. Chem. 2003; 278:26311-26314). The matrix metalloproteinase BMP-1 was shown to more rapidly become secreted when the furin cleavage site RSRR in its prodomain was mutated to RSAA (Leighton and Kadler J. Biol. Chem. 2003: 278:18478-18484). When the furin cleavage site for nerve growth factor (NGF) was mutated cleavage occurred at an alternate site but the alternate NGF did not undergo regulated secretion (Lim et al. BBRC 2007; 361:599-604).
The pro-domain of the TGF-β family members, including all BMPs, is believed to have several functions. It appears to be required for the folding, dimerization and secretion of mature active TGF-β and activin (Gray & Mason. Science 1990; 247:1328-1330). Further, in the case of TGF-β, continued association of the N-terminal and C-terminal domain after proteolytic cleavage renders the complex inactive or latent (Gentry et al. Biochemistry 1990; 29:6851-6857). ProBMP-4 has been reported to be biologically inactive (Cui et al. EMBO J. 1998; 17:4735-4743), although E. coli produced proBMP-2 has been reported to posses biological activity (Hillger et al. J. Biol. Chem. 2005; 280:14974-14980) and CHO cell produced rh-proBMP-9 has similar activity as mature rhBMP-9 in various in vitro assays (Brown et al. J. Biol. Chem. 2005; 280:25111-25118).
Comparison of the production, processing and secretion of mouse and human BMP-15 produced by transfected HEK293 cells indicated that human BMP-15 (hBMP-15) was secreted into the conditioned medium; however mouse BMP-15 (mBMP-15) was not secreted. Unlike hBMP-15, mBMP-15 pro-protein is not cleaved into a mature protein after proteolytic processing, but is targeted for intracellular degradation. When the hBMP-15 pro-domain was fused with the mature region of mBMP-15, there was secretion of mBMP-15 mature protein into the conditioned cell culture media (Hashimoto, et al. Proc. Natl. Acad. Sci. 2005; 102: 5426-543). Thus, in the case of BMP-15 at least, it appears that the proper processing of the pro-protein is significant for the secretion of the mature proteins.
When a Val residue is exchanged to a Gly at AA position 130 in the pro-domain of BMP-7 normal levels of the precursors and mature protein were found in the Xenopus oocyte lysates, indicating that stability and processing of the precursor are not affected by the mutation. However, there was a dramatically reduced amount of both the pro-domain peptide and the mature protein in the conditioned medium (Dick, et al. Development, 2000; 127: 343-354). In-frame deletion of the pro-peptide of BMP-2 yielded a polypeptide that was not secreted from the cell, suggesting that the pro-peptide may therefore be involved in processing and secretion of mature BMP-2 protein (Israel et al. Growth Factors 1992; 7: 139-150). A hybrid of the pro-domain of BMP-2 fused to the mature region of BMP-4 has been constructed and shown to secrete mature biologically active BMP-4 at an enhanced level (Hammonds et al. Mol Endocrinol 1991; 5: 149-155). These results indicate that the prodomain plays an important role in the folding and secretion of proteins.
Limited endoproteolysis of the prodomain of a protein is a general mechanism generating a diversity of biologically active peptides and proteins in all eukaryotic phyla. This is performed by a small number of Ca(2+)-dependent serine proteases collectively called proprotein convertases (PCs) (for reviews see Seidah & Chretien Curr Opin Biotechnol 1997; 8:602-607, Taylor et al. FASEB J 2003; 17:1215-1227). These PC possess homology to the endoproteases subtilisin (bacteria) and kexin (yeast). This family of mammalians PCs is currently comprised of furin (also called paired basic amino-acid-cleaving enzyme (PACE)), PC1/PC3, PC2, PC4, PACE4, PC5/PC5A/PC6, PC5B/PC6B (a spice variant of PC5A) and PC7/PC8/lymphoma proprotein convertase. They share a high degree of amino-acid identity of 50-75% within their catalytic domains. Furin and PC7 are expressed ubiquitously, PACE-4, PC5A and PC5B are expressed at varying levels in many tissues while PC1, PC2, and PC4 are restricted to specific tissues (Dubois et al. Am. J. Path. 2001; 158:305-616).
One of the major recognition motifs for these enzymes involves cleavage at either specific single or pairs of basic residues of the general formula (R/K)-Xn-(R/K), where n=0, 2, 4 or 6. Such sites are found in a variety of protein precursors in all eukaryotes, including those of endocrine and neural polypeptide hormones (including PTH, Insulin), enzymes (including furin, MMP-1, MMP-13), growth factors (including TGF-β1, BMP-2, BMP-4, BMP-7, PDGF, IGF-1, IGF-2, VEGF, FGF-23, EGF, PTHrP), receptors, adhesion molecules (including many integrins), viral glycoproteins, coagulation factors and even cell signaling molecules (see Seidah & Chretien Curr Opin Biotechnol 1997; 8:602-607, Khatib et al. Am J Pathol 2002; 160: 1921-1935, Taylor et al. FASEB J 2003; 17:1215-1227).
Both BMP-2 and BMP-4 posses 2 PC recognition sites, called the S1 (AA 278) and S2 (AA 245) sites, while BMP-7 possess only one recognition site, the S2 site (Sopory et al. J Biol. Chem. 2006; 281:34021-34031). ProBMP-4 is cleaved first at the S1 site to produce the mature BMP-4 molecule and the pro-domain. The prodomain associates with the mature protein non-covalently until it is cleaved at the S2 site. While the prodomain remains associated with the mature protein the complex is targeted for intracellular degradation (Degnin et al. Mo Biol. Cell 2004; 15:5012-5020.). Further it has been shown that mutation of the S2 site results in tissue specific loss of BMP-4 activity (Goldman et al. Development 2006; 133:1933-1942). Both the prodomain and mature BMP-2 are secreted into conditioned medium of CHO cells expressing proBMP-2, however approximately 5 times more prodomain than mature protein was detected in the medium (Israel et al. Growth Factors 1992; 7:130-150).
ProBMP-4 can be cleaved by furin, PC6, PC7 and PACE 4 in vitro while in vivo studies suggest that either furin, PC6, and or PACE 4 is the PC responsible for cleavage intracellularly (Cui et al. EMBO J. 1998; 17: 4735-4743, Tsuji et al. J. Biochem 1999; 126:591-603). Studies on the efficiency of the different PC to cleave proTGF-β1 in cell systems identified the order of activity from most to least as furin>PC5B=PACE-4>PC7>PC-1 while PC2 and PC5A had little effect on the proTGF-β1 protein (Dubois et al. Am. J. Path. 2001; 158:305-616).
The human colon carcinoma LoVo cell line possesses a point mutation in the furin gene (fur) which results in LoVo cells completely lacking furin enzymatic activity (Takahashi et al. Biochem. Biophys. Res. Comm. 1993; 195:1019-1026). When these cells are transfected with TGF-β1 they secrete only proTGF-β1 which is inactive (Dubois et al. J. Biol. Chem. 1995; 270:10618-10624). When these cells are co-transfected with various proprotein convertases, the degree of cleavage of proTGF-β1 varied depending on the PC co-transfected with the TGF-β1 (Dubois et al. Am. J. Path. 2001; 158:305-616).
A CHO-derived cell line that over-expresses furin when transfected with cDNA coding for full length TGF-β1 or von Willebrand Factor (vWF) demonstrated increased production of active TGF-β1 or vWF (Ayoubi et al. Mol. Biol. Rep. 1996; 23:87-95), however no effort was made to determine whether the total amount of recombinant protein produced (active+inactive) was more or less than in CHO cells not over-expressing furin. When α1-antitrypsin Portland (α1-PDX), an inhibitor of furin was ectopically expressed in Xenopus embryos it blocked BMP-4 activity upstream of the receptor.
Israel and co-workers attempted to enhance the amount of mature BMP produced in CHO cells by using protease inhibitors, however they stated that they “were unable to increase the amount of BMP-2 mature protein by including a large number of different protease inhibitors in the culture medium.” They did not report which inhibitors they tested (Israel et al. Growth Factors 1992; 7: 139-150).
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.